Cytochrome oxidase staining reveals functionally important activity bands in the olfactory epithelium of newborn rat

Abstract

We used cytochrome oxidase (CytOx) staining intensity, which is correlated with neuronal functional activity, to evaluate maturity and functionality of newborn rat olfactory epithelium (OE) and olfactory receptor neurons (ORNs). Nasal olfactory tissue of neonatal rats was stained with CytOx and analyzed qualitatively and quantitatively. Results revealed that newborn OE shows six differentially stained horizontal bands. Bands run parallel to the OE surface and were categorized as very light, medium or darkly stained. A narrow and pale Band 1 overlapped with horizontal basal cells. Next, a wide and lightly stained Band 2 was observed that coincides with the globose basal cell layer and immature ORNs, deep in OE. Next apically, a medium-staining Band 3 overlapped with ORN perikarya. Closer to the surface, a medium to light Band 4 was discerned where dendrites of mature ORNs normally occur. This band was interrupted with lighter areas due to the presence of supporting cells nuclei. Next, a superficial but dark Band 5 occurred, populated by the apical portions of ORN dendrites and their ciliated knobs and by supporting cell apices; mitochondria in apices of supporting cells contribute predominantly to dense staining of this Band 5. Apical to Band 5, a thin and fairly light Band 6 was observed which overlaps with the mucus layer that contains part of the ORN knobs, their cilia and supporting cell microvilli. Along the length of ORN dendrites, apical segments just below the ORN knobs, and wide basal segments showed a darker staining than the middle segments implying "microzones" of higher neural activity within the most apical and basal regions of dendrites. Our findings agree with ultrastructural studies showing a presence of mitochondria in knobs, basal portions of ORN dendrites and in OE supporting cell apices, suggesting that apical regions of both olfactory and supporting cells near the surfaces are metabolically most active, in odorant detection, signal processing, and detoxification, the latter for supporting cells.

Proteomic Analysis of a Membrane Preparation from Rat Olfactory Sensory Cilia

“…Ezrin, for example, a cytoskeletal protein expressed in supporting cells but not in ORNs (Elsaesser et al 2005; GFP + /GFP ÿ ratio = 0.1), points to the presence of small amounts of microvillar material in the preparation. In addition, the xenobiotic-metabolizing enzymes are thought to be expressed in ORNs and epithelial supporting cells but with much higher expression levels in the latter (Dahl and Hadley 1991;Thornton-Manning and Dahl 1997;Pataramekin and Meisami 2005). Nevertheless, in conjunction with the immunoblot experiments it could be stated that the preparation is strongly enriched in ORN membranes and the contribution of other cell types appears to be small.…”

“…Moreover, the constant exposure of the cilia to environmental compounds may require an effective xenobiotic biotransformation metabolism, which protects the olfactory epithelium and the ORNs from cytotoxic com-pounds and terminates the sensory response (reviews: Marini et al 1998;Ling et al 2004). The expression of xenobioticmetabolizing enzymes in ORNs is supported by the finding that cytochrome oxidase staining, which is correlated with xenobiotic functional activity, was not only observed in epithelial supporting cells but also in ORN perikarya, dendrites and their ciliated knobs, as well as within the mucus layer that contains parts of the ORN knobs, their cilia, and supporting cell microvilli (Pataramekin and Meisami 2005). However, highest activity levels of xenobiotic-metabolizing enzymes were generally found in supporting cells, suggesting that protection of the entire sensory neuroepithelium from environmental compounds is mainly a task of supporting cells (reviews: Dahl and Hadley 1991;Thornton-Manning and Dahl 1997;Marini et al 1998).…”